Over the past few years, we have constructed large combinatorial chemical libraries (>100,000 diverse and discrete small molecules) and carried out high throughput phenotypic screens of these libraries to identify small molecules that can control stem cell fate in several systems. Progresses in developmental and beta-cell biology have suggested that the in vitro generation of insulin-producing beta-cells from pluripotent embryonic stem (ES) cells will require a stepwise differentiation process involving at least five steps: ES cells -> mesendoderm/primitive streak -> definitive endodermal cells -> pancreas -> endocrine precursors -> islet (including mature insulin-producing beta-cells). Correspondingly, we propose to develop cell-based, phenotypic assays, which will be implemented into high throughput screens of 100,000 discrete and diverse compounds to identify small molecules that can regulate the important early steps of beta-cell development in human ES (hES) cells (WA-01). Specifically, we will screen for chemical compounds that can differentiate hES cells selectively into mesendoderm and definitive endoderm using lineage specific bacterial artificial chromosome (BAC) transgenic reporter hES cell lines. We will further confirm and characterize the effects and activities of selected compounds via various in-depth cellular/biochemical assays, and carry out structure-activity-relationship (SAR) studies of the selected hit compounds to optimize their potency and specificity. Collectively, the studies described in this proposal will generate new chemical tools for selectively and efficiently producing lineage-specific progenitor cells toward pancreatic beta cells for various applications, broaden our understanding of the developmental signaling pathways regulating such early-stage cellular specification, and may ultimately facilitate development of small molecule therapeutics to treat type 1 diabetes. [unreadable] [unreadable] [unreadable]